Korean J Clin Pathol.  1997 Feb;17(1):99-108.

The Polymerase Chain Reaction Applying dUTP-UDG Protocol for Detection of Mycobacterium tuberculosis

• Jeong OY
• Jang SJ
• Yeam YS
• Park YJ
• Lee SI
• Kim YS

Abstract

BACKGROUND: The polymerase chain reaction(PCR) assay is rapid, sensitive analytical technique but has problem of high false-positive rate. We applied dUTP-UDG PCR (dU-PCR) method to prevent carryover contamination major source of high false positive in PCR assays, for detection of Mycobacterium tuberculosis.
METHODS
The PCRs for detection of M. tuberculosis were performed with P1 and P2 primers based on IS6110 repeated sequence. FTC-2000 was used for capillary PCR and Uno-Thermoblock was used for heating block PCR. In order to evaluate the effect of dU-PCR controlling carryover contamination, PCRs were performed in the presence of UDG and the absence of UDG. To compare the sensitivity of usual dT-PCR with dU-PCR, chromosomal DNA of M. tuberculosis ranging 500pg to 0.5fg were amplified by dT-PCR and dU-PCR method using two different thermocycler, capillary and heating block type, respectively. RESULT: The dU-PCR using UDG prevented carryover contamination by amplicon DNA up to 500pg. By capillay PCR method, the lower limits of detectability of dT-PCR and dU-PCR were 0.5fg and 500fg, respectively, which indicates the sensitivity of dU-PCR was lower than dT-PCR. But by heating block method, the lower limits of detectability of both method of dU and dT-PCR were 0.5fg. So the sensitivity of dU-PCR was same as dT-PCR.
CONCLUSION
The dU-PCR by heating-block method was sensitive test for detection of M. tuberculosis that effectively prevent carryover contamination by amplicon.

MeSH Terms

Capillaries
DNA
Heating
Hot Temperature
Mycobacterium tuberculosis*
Mycobacterium*
Polymerase Chain Reaction*
Tuberculosis
DNA